New direct-acting antivirals against hepatitis C virus (HCV) cure the virus in most patients. However, these compounds are expensive and not available to most of the 170 million people that are worldwide infected with HCV. There are also no approved vaccines or anti-viral compounds for other members of the flaviviridae, such as Dengue virus, West Nile virus or Zika virus. Thus, identification of cellular genes that are essential for virus propagation is highly significant, because such targets are not regulated by the error-prone viral RNA polymerase and, therefore, offer a high barrier to resistance. The long-term goal of this application is to explore the mechanism by which certain noncoding RNAs, such as microRNAs and circular RNAs, display pro-viral or anti-viral activities. It is known that microRNA miR-122 attaches as an oligomeric complex to the 5? end of the viral genome and protects it from degradation by host exonucleases. Curiously, escape mutants that harbor a single C3U mutation in the viral genome were detected in the serum of 122-antagomir treated patients. The overall objective of the first aim to is identify interactions of between C3U HCV genome with nucleic acids or proteins that allow the virus to persist when miR-122 abundance is reduced. The central hypothesis is that novel RNA-RNA or protein RNA interactions in the C3U HCV genome allow stabilization and expression of the viral genome in the cells during reduced miR-122 abundances. Novel cell-based protein biotinylation assays and approaches that detect tertiary RNA structures will be used to study RNA-protein and RNA-RNA interactions in wildtype and mutant viral RNAs in cultured liver cells. Steps in the viral life cycle that are modulated by such nucleic acid-protein interactions will be identified. The overall objective of the second aim is based upon the finding that the host cell-derived circular RNA (cRNAs) landscape is altered during HCV infection. The potential mechanisms by which cRNAs exert their pro- and antiviral functions will be explored. First, effects of cRNA-mediated sequestration of proteins and microRNAs on HCV RNA amplification will be examined. Because methylation of specific adenosines in cRNAs has been shown to induce translation initiation in cRNAs, the methylation status in cRNAs will be determined. The properties of methylated cRNAs to synthesize small peptides will be examined using genetic and proteomic approaches. Overall, the application details innovative paradigm-shifting concepts to study roles for noncoding RNAs in virus-host interactions, using novel detection methodologies. The rationale for this proposal is that noncoding RNAs, such as microRNAs and cRNAs, affect HCV pathogenesis. It has been shown that microRNAs can be targeted in HCV patients, resulting in loss of viral RNA abundance. Thus, targeting pro-viral cRNAs in the liver offers a novel antiviral strategy.